Hepatitis C virus (HCV) infection is associated with a delayed and inadequate humoral immune response, and also with an autoimmune condition, mixed cryoglobulinemia (MC). MC patients are at increased risk for development of B cell non-Hodgkin lymphoma. We found that patients with MC have clonal or oligoclonal expansions of B cells expressing rheumatoid factor (RF)-like IgM, frequently encoded by VH1-69, JH4, and V:3-20 Ig gene segments. This IgM's RF activity arises as a result of somatic hypermutation. The RFs are re- lated to broadly active neutralizing antibodies observed in patients responding to HIV, influenza, and HCV infections. We propose that B cells bearing VH1-69 and V:3-20-coded IgM are activated by recognition of a viral antigen, and that polyreactivity of these IgMs may contribute to their ability control HCV infection. However, somatic hypermutation renders these antibodies autoreactive and limits their efficacy in vivo. A major fraction of the RF-bearing B cells in the blood of HCV+MC+ patients is anergic. In this application, we propose to test this model by (Aim 1) defining the repertoire of HCV-reactive antibodies in HCV patients with and without MC; (Aim 2) testing the hypothesis that VH1-69-coded antibodies react to lipid components of the HCV virion; and (Aim 3) defining the activation requirements for anergic RF-bearing B cells in HCV+MC+ patients. For Aim 1, we will prepare long-lived B cell lines from HCV patients with and without MC. B cells will be selected for reactivity to specific HCV protein antigens, or for expression of VH1-69 immunoglobulin. B cell lines expressing HCV- specific or HCV-neutralizing antibody will be characterized by cloning and sequencing of the rearranged expressed immunoglobulin genes. This will permit us to define the immunoglobulin repertoire associated with reactivity to HCV. For Aim 2, we will take advantage of a panel of RFs from HCV+MC+ patients that we have already characterized. These RFs are expressed in transfected cells as pentameric human IgM. Both the somatically-hypermutated and non-mutated (germline counterpart) RFs are available for study. We will evaluate the ability of these IgMs to bind to a panel of lipid moieties. For Aim 3, we will test the hypothesis that the anergic behavior of RF-bearing B cells in vitro represents a homeostatic mechanism to limit autoantibody production; we propose that HCV RNA present in immune complexes serves as a potent signal to drive the proliferation and differentiation of RF-bearing B cells. This mechanism will be tested by in vitro stimulation of RF-bearing B cells with a panel of stimuli including cell culture-produced HCV, immune complexes built from HCV and anti-HCV IgG, cytokines, and T cell help. Outcomes to be measured include B cell Ca++ mobilization, proliferation, and antibody production. These studies will provide new insights into the pathogenesis of MC and the mechanisms that may limit generation of effective HCV-specific immune responses. Our group is uniquely qualified to perform this study because of our expertise in HCV immunology and virology, and our ready access to viruses, viral proteins, and patients for study. !